Skeletal muscle is important for drug and toxicity testing given the relative size of the muscle mass and cardiac output that passes through muscle beds, the key role of muscle in energy substrate metabolism and diabetes, its role in mediating the severity of peripheral arterial disease and heart failure, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To develop a system for functional and drug testing under physiological conditions, we will incorporate three-dimensional skeletal muscle cultures in a circulatory system that consists of a high-pressure arterial system that carries media to various tissue microcirculatory organ beds and returned via a low-pressure venous system. Arterial vessels will consist of an inner layer of endothelium and layers of differentiated vascular smooth muscle cells or mesenchymal stem cells. A computer controlled pump and valve system will pump small volumes of fluid to mimic arterial flow. Measurement of O2, CO2 and pressure will be used to control flow to the various beds. The modular design of the microcirculatory organ beds facilitates integration with a broad array of other organ and tissue mimics as part of the UH3 phase of the Cooperative Agreement. All experiments will use primary human cells. To generalize the applicability of the test bed, we will develop mature smooth muscle cells and skeletal muscle from iPS cells. In Aim 1, we will fabricate and test a branching network of small caliber blood vessels consisting of several layers of contractile human smooth muscle cells or mesenchymal stem cells and a confluent layer of endothelium. Flow rates, vessel distension and contraction, and the resistance of the microfluidic microcirculatory beds lined with endothelium will control the flow distribution to the different microcirculatory beds. In Aim 2, we will develop three-dimensional constructs of skeletal muscle and fibroblasts under tension. Different levels of oxygen partial pressure in the arterial and venous inflow lines will be used to produce a range of oxygen gradients. The muscle will be connected to posts containing a ferrogel that contracts under electromagnetic stimulation and thereby loads the muscle fibers. Endothelium will cover the outside of the three-dimensional muscle cultures, serving as an interface between the perfusion medium and skeletal muscle fibers. Oxygen gradients across the muscle layer will be controlled by cell density and thickness of the cell layer. We will fabricate an electrode system to electrically stimulate the fibers and measure force production. In Aim 3, we will combine the vascular and muscle units and run the unit for four weeks. Measurement of O2, CO2 and pressure will be used to control overall flow and regulate flow to the different beds. We will assess vessel dilation and muscle function. In Aim 4, the completed system will be used to test the effect of local release of vasodilators and vasoconstrictors on flow distribution, glucose metabolism and oxygen uptake by muscle. We will examine the response of blood vessels and muscle to an inflammatory stimulus. Metabolic profiling will be performed to simulate different physiological conditions and response to drugs and toxins. PUBLIC HEALTH RELEVANCE: This project will provide new methodology to examine the function of human skeletal muscle in the laboratory and to test the effectiveness and toxicity of drugs. We expect that the results of this project will facilitate the screening of candidate drugs o treat disorders of skeletal muscle and blood vessels.